Expression of the ompC and ompF genes coding for the major outer membrane proteins, OmpC and OmpF, respectively, is known to be controlled by at least two regulatory genes, ompR and envZ, which together comprise a single ompB operon. We constructed chromosomal mutants with either ompR-envZ deletion or envZ deletion. Characterization of these deletion strains showed that the OmpR protein is necessary for transcription of the ompC and ompF genes, and the EnvZ protein is essential for normal regulation of the ompC and ompF expression, which is affected by the medium osmolarity. We also constructed several plasmids carrying different portions of the ompB operon. Characterization of these plasmids allowed us to identify the OmpR protein with an apparent molecular weight of 29 kilodaltons (kDa) and the EnvZ protein with an apparent molecular weight of 50 kDa. The initiation codon for EnvZ translation appeared to overlap with the termination codon for OmpR translation. It was also found that a truncated EnvZ polypeptide (44 kDa) which lacks the N-terminal 55 amino acid residues can complement the envZ deletion mutant. Based on these results, the structure and function of the ompB operon are discussed in relation to the regulation of ompC and ompF expression.